Dental caries is a disease of humans and animals, which is primarily induced on the surface of a tooth by the action of cariogenic oral bacilli and progressively breaks down its structure. Among various cariogenic oral bacilli, Streptococcus mutans is believed to be the most important. It has previously been reported, for example, in Bergy's Manual of Determinative Bacteriology, page 502 (1974) that a relationship exists between dental caries and S. mutans. It is suggested that S. mutans is a similar organism to S. salivarius, even though S. mutans has not yet been extensively studied and compared with S. salivarius, Nowadays, however, typical oral streptococci are clearly distinguished from each other, for example, with reference to the following characteristics:- (a) S. mutans decomposes mannitol and sorbitol and also produces both water-soluble and insoluble dextran-like polysaccharides (hereinafter referred to as DPS) from sucrose. (b) S. sanguis produces from sucrose water-soluble DPS having a weaker adhering ability and also produces ammonia from arginine; and (c) S. salivarius produces fructans from sucrose.
As is well known, insoluble DPS produced by S. mutans adhere to the surface of teeth to form dental plaque. The major portion of S. mutans in the oral floras resides in the plaque and produces lactic acid which is believed to break down the teeth. Other cariogenic oral bacilli also produce lactic acid. However, the lactic acid produced by S. mutans is not released from the plaque, but directly accumulates on the surface of the tooth.
Strains of S. mutans are classified according to the classification by Bratthal et al into 7 serological groups (serotypes), designated "a" to "g", in view of the immunological specificities of their cell wall antigens (Bratthal. Odont. Rev. 20:141, 1970 and ibid., 20:23, 1970 and Perch et al, Acta. Path. Microbiol. Scand. Section B, 82:357, 1974). A further classification scheme for classification of S. mutans has been proposed by Makoto Sato. According to this classification scheme, strains of S. mutans are classified into Human I (HI), Human II (HII) and Rat (R) types. The following has been reported by Sato:
93.9% of S. mutans of human origin were Human I type and the rest being Human II type; PA0 (b) all strains of rat origin were Rat type; PA0 (c) no S. mutans was isolated from mice and guinea pigs; PA0 (d) all strains originating from hamsters and monkeys were Human I type; PA0 (e) Human I, Human II and Rat types correspond to "c, e and f", "d and g", and "a and b" serotypes of Bratthal's classification (J. of Dental Health, Vol. 28, No. 2, pages 100-123 1978 in the Japanese version). PA0 (1) A PLS antigen is an acidic protein containing about 15-25% (e.g. about 20%) protein and about 75-85% (e.g. about 80%) carbohydrates and having a molecular weight of about 60,000-90,000 (e.g. ca. 75,000). PA0 (2) Polyacrylamide disc electrophoresis of a PLS antigen results in a characteristics broad band towards the cathode. PA0 (3) If sucrose density-gradient ultracentrifugation is carried out with a PLS antigen, the antigen is found in the fractions having a sucrose density of about 10-20% and a specific gravity of about 1.3 to 1.4. PA0 (4) If gel filtration of a PLS antigen is carried out using Sephadex G-100 (Pharmacia Fine Chemicals AB., Sweden), two peaks are eluted (monitored by absorption at 280 nm). The PLS antigen is recovered from the first peak, of which reactivity with human or rabbit serum is positive. PA0 (5) A PI value for a PLS antigen of not more than about 3.5 may be obtained by the electric focusing method according to Versterberg et al method using 1% of the carrier ampholite (pH 3.0-10.0; commercial product of LKB Produkter AB., Sweden).
Possible approaches for inhibiting human dental caries induced by S. mutans by immunological means have previously been put forward. British Patent 1,375,866, for example, discloses a dental vaccine which comprises as antigen at least a part of the cell of a cariogenic strain of S. mutans SSC, of which characteristics are the same as the characteristics of Streptococcus mutans NCTC 10449 which is a well-recognized representative cariogenic strain present in the oral cavity of humans. This patent discloses as preferred antigens, the cell wall and the enzyme from the cultured broth. All vaccines described in its examples use, as antigen, the whole cells inactivated with formalin or phenol. However, it is well known that administration of whole cell antigen originating from S. mutans to animals is liable to cause various undesirable side effects such as, for example, cross-reaction with the heart muscle antigen, allergic reaction and the like. To our knowledge, it has not yet been reported that such a vaccine has been used to inhibit human dental caries induced by S. mutans with good results.
British Patent No. 1,505,513 and U.S. Pat. No. 4,324,784 disclose cow's milk containing antibodies for inhibiting S. mutans, produced by immunizing a cow with heat-inactivated whole cells of particular strains of S. mutans viz. S. mutans AHT, BHT, 10449 and 6715 respectively of the serotypes of "a", "b", "c" and "d". However, the known cow's milk cannot be considered suitable for human use, since the cow's milk lacks antibodies capable of inhibiting S. mutans 10449 (serotype "c"; Human I type) which is well recognized as a representative cariogenic strain in the oral cavity of humans or any other strains of Human I type S. mutans, which represents more than 90% of cariogenic strains in the oral cavity of humans, even though the cow's milk is more or less active upon S. mutans 6715 (serotype "d").
U.S. Pat. No. 4,442,085 (Colman) discloses that Antigen A capable of inhibiting S. mutans and having a molecular weight of 32,000-26,000 and an isoelectric point of 4.3 is present in the cell walls and cultures of strains of S. mutans of serotypes "c, e and f". Antigen A may be extracted from the cell wall by a boiling aqueous solution of SDS(sodium dodecyl sulfate); a detergent followed by chromatography of the resultant precipitate for purification. However, it is not clear whether Antigen A is active upon Human II type strains (serotypes "d and g").
U.S. Pat. No. 4,521,513 (Russell) discloses that Antigen C capable of inhibiting S. mutans and having a molecular weight of 65,000-75,000 and an isoelectric point of 4.21 to 4.69 is present in the cell walls and cultures of strains of S. mutans of serotypes "c, e and f". Antigen C may be separated from the cell extract with a boiling solution of sodium dodecyl sulphate (detergent) and purified by chromatography., or from the culture filtrate and cell extract by chromatography. However, it is not clear whether Antigen C is active upon Human II type strains of S. mutans.
U.S. Pat. No. 4,150,116 (Taubman et al) discloses that purified glucosyltransferase (GFT) is capable of inhibiting S. mutans, which may be recovered from cellular polysaccaride with a denaturing agent and purified by chromatography.
Bowen et al (British Dent. J. Vol. 139, pp. 45-58, 1975) disclose that various antigens originating from strains of S. mutans such as antigens from the whole cells, broken cells and glucosyltransferase etc. may be used for immunizing animals preferably by intra-mucosal injection.
Meanwhile, it was previously known that strains of S. mutans have the pili-like structures (fimbriae) on the surface of the cell wall, by means of which they adhere (infect) to the oral cavity of humans and animals (Gibbons et al, Ann. Rev. Microbiol., 29:19-44, 1975). However, it has not yet been reported in the art that antigen originating from such pili-like structures of S. mutans is capable of specifically inhibiting or preventing the adherence (infection) of strains of S. mutans to the oral cavity of humans.
The present invention is based upon the discovery that antigens which we have isolated from the pili-like structures (PLS) of strains of Human type S. mutans are capable of inhibiting the adherence (infection) of strains of Human type S. mutans to the oral cavity of humans with excellent results and that the desired antigens may be extracted by using a hypertonic buffer solution under mild conditions.